Affiliation: Department of Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America. halford@siumed.edu

ABSTRACTWe lack a correlate of immunity to herpes simplex virus 2 (HSV-2) that may be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes. This gap in knowledge is often attributed to a failure to measure the correct component of the adaptive immune response to HSV-2. However, efforts to identify a correlate of immunity have focused on subunit vaccines that contain less than 3% of HSV-2's 40,000-amino-acid proteome. We were interested to determine if a correlate of immunity might be more readily identified if 1. animals were immunized with a polyvalent immunogen such as a live virus and/or 2. the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2's antigens (pan-HSV-2 IgG). Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0 (-) viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.

pone-0065523-g004: Pan-HSV-2 IgG levels correlate with protection against vaginal HSV-2 challenge in guinea pigs.(A) Design of guinea pig vaccine-challenge experiment. Guinea pigs were immunized in their right, rear footpads on Day 0 with gD-2, culture medium (mock), HSV-2 0ΔNLS, or HSV-2 MS, as described in the Results (n = 5 per group). Guinea pigs immunized with HSV-2 MS received 1 mg/ml acyclovir in drinking water from Days 0 to 20 post-immunization to restrain the pathogenesis of a primary exposure to wild-type HSV-2. All guinea pigs were boosted in their left, rear footpads on Day 30 with an equivalent, booster immunization; MS-immunized guinea pigs did not receive acyclovir during the secondary boost. On Day 75, blood was harvested, and on Day 90, guinea pigs were challenged with 2×106 pfu per vagina of wild-type HSV-2 MS. (B) Mean ± sem pfu of HSV-2 shed per vagina between Days 1 and 8 post-challenge in guinea pigs that were naïve (n = 5) or were immunized with gD-2+ alum/MPL (n = 4), HSV-2 0ΔNLS (n = 5), or an acyclovir (ACV)-restrained HSV-2 MS infection (n = 5). A single asterisk (*) denotes p<0.05 and a double asterisk (**) denotes p<0.0001 that HSV-2 MS vaginal shedding was equivalent to naïve guinea pigs on that day, as determined by one-way ANOVA and Tukey’s post hoc t-test. (C) For each guinea pig (one symbol per animal), the average amount of infectious HSV-2 shed on Days 1, 2, 3, 4, 6, and 8 post-vaginal challenge (y-axis) was plotted as a function of pre-challenge pan-HSV-2 IgG levels observed in the same guinea pig (x-axis). The solid black line represents the best-fit linear regression model, y = 3.77–0.95x, for these 19 matched datum pairs. (D) Mean ± sem of log (pan-HSV-2 IgG) in each immunization group is plotted on the x-axis versus mean ± sem vaginal HSV-2 shedding on the y-axis. The solid black line represents the best-fit linear regression model, y = 3.77–0.95x, for these 4 matched averages (r2 = 0.98). Groups of immunized guinea pigs that exhibited a significant reduction in vaginal HSV-2 shedding relative to naïve guinea pigs are indicated by a single asterisk (*; p<0.05) or double-asterisk (**; p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test. (E) The worst case of perivaginal disease in each group of naïve or immunized guinea pigs on Day 7 post-challenge. Survival frequency refers to the frequency with which animals in each immunization group survived until Day 30 post-challenge.

Mentions:
A third, prospective analysis was performed to determine if pre-challenge pan-HSV-2 IgG levels varied in proportion to protection against HSV-2 in a species other than mice. To address this question, groups of n = 5 guinea pigs were immunized on Days 0 and 30 in their right and left rear footpads, respectively, with 1. culture medium (naïve), 2. 5 µg gD-2 adjuvanted with alum and 20 µg MPL, 3. 2×106 pfu HSV-2 0ΔNLS, or 4. 2×106 pfu of wild-type HSV-2 MS where acyclovir was used to restrict the pathogenesis of the primary exposure to MS (Fig. 4A). Guinea pigs were bled on Day 75 and challenged on Day 90 with 2×106 pfu HSV-2 MS per vagina (Fig. 4A). Unfortunately, one gD-2-immunized guinea pig was lost to an anesthetic overdose; thus, only n = 4 gD-2-immunized guinea pigs were available following HSV-2 vaginal challenge. Naïve guinea pigs shed peak titers of ∼200,000 pfu per vagina on Day 2 post-challenge (Fig. 4B). Guinea pigs immunized with gD-2 shed an average 5-fold less HSV-2 relative to naïve guinea pigs between Days 1 and 8 post-challenge (Fig. 4B). In contrast, guinea pigs immunized with HSV-2 MS or 0ΔNLS shed an average 150- and 200-fold less HSV-2, respectively, relative to naïve guinea pigs (Fig. 4B).

pone-0065523-g004: Pan-HSV-2 IgG levels correlate with protection against vaginal HSV-2 challenge in guinea pigs.(A) Design of guinea pig vaccine-challenge experiment. Guinea pigs were immunized in their right, rear footpads on Day 0 with gD-2, culture medium (mock), HSV-2 0ΔNLS, or HSV-2 MS, as described in the Results (n = 5 per group). Guinea pigs immunized with HSV-2 MS received 1 mg/ml acyclovir in drinking water from Days 0 to 20 post-immunization to restrain the pathogenesis of a primary exposure to wild-type HSV-2. All guinea pigs were boosted in their left, rear footpads on Day 30 with an equivalent, booster immunization; MS-immunized guinea pigs did not receive acyclovir during the secondary boost. On Day 75, blood was harvested, and on Day 90, guinea pigs were challenged with 2×106 pfu per vagina of wild-type HSV-2 MS. (B) Mean ± sem pfu of HSV-2 shed per vagina between Days 1 and 8 post-challenge in guinea pigs that were naïve (n = 5) or were immunized with gD-2+ alum/MPL (n = 4), HSV-2 0ΔNLS (n = 5), or an acyclovir (ACV)-restrained HSV-2 MS infection (n = 5). A single asterisk (*) denotes p<0.05 and a double asterisk (**) denotes p<0.0001 that HSV-2 MS vaginal shedding was equivalent to naïve guinea pigs on that day, as determined by one-way ANOVA and Tukey’s post hoc t-test. (C) For each guinea pig (one symbol per animal), the average amount of infectious HSV-2 shed on Days 1, 2, 3, 4, 6, and 8 post-vaginal challenge (y-axis) was plotted as a function of pre-challenge pan-HSV-2 IgG levels observed in the same guinea pig (x-axis). The solid black line represents the best-fit linear regression model, y = 3.77–0.95x, for these 19 matched datum pairs. (D) Mean ± sem of log (pan-HSV-2 IgG) in each immunization group is plotted on the x-axis versus mean ± sem vaginal HSV-2 shedding on the y-axis. The solid black line represents the best-fit linear regression model, y = 3.77–0.95x, for these 4 matched averages (r2 = 0.98). Groups of immunized guinea pigs that exhibited a significant reduction in vaginal HSV-2 shedding relative to naïve guinea pigs are indicated by a single asterisk (*; p<0.05) or double-asterisk (**; p<0.001), as determined by one-way ANOVA and Tukey's post-hoc t-test. (E) The worst case of perivaginal disease in each group of naïve or immunized guinea pigs on Day 7 post-challenge. Survival frequency refers to the frequency with which animals in each immunization group survived until Day 30 post-challenge.

Mentions:
A third, prospective analysis was performed to determine if pre-challenge pan-HSV-2 IgG levels varied in proportion to protection against HSV-2 in a species other than mice. To address this question, groups of n = 5 guinea pigs were immunized on Days 0 and 30 in their right and left rear footpads, respectively, with 1. culture medium (naïve), 2. 5 µg gD-2 adjuvanted with alum and 20 µg MPL, 3. 2×106 pfu HSV-2 0ΔNLS, or 4. 2×106 pfu of wild-type HSV-2 MS where acyclovir was used to restrict the pathogenesis of the primary exposure to MS (Fig. 4A). Guinea pigs were bled on Day 75 and challenged on Day 90 with 2×106 pfu HSV-2 MS per vagina (Fig. 4A). Unfortunately, one gD-2-immunized guinea pig was lost to an anesthetic overdose; thus, only n = 4 gD-2-immunized guinea pigs were available following HSV-2 vaginal challenge. Naïve guinea pigs shed peak titers of ∼200,000 pfu per vagina on Day 2 post-challenge (Fig. 4B). Guinea pigs immunized with gD-2 shed an average 5-fold less HSV-2 relative to naïve guinea pigs between Days 1 and 8 post-challenge (Fig. 4B). In contrast, guinea pigs immunized with HSV-2 MS or 0ΔNLS shed an average 150- and 200-fold less HSV-2, respectively, relative to naïve guinea pigs (Fig. 4B).

Bottom Line:
These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range.For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge.Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.

Affiliation:
Department of Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America. halford@siumed.edu

ABSTRACTWe lack a correlate of immunity to herpes simplex virus 2 (HSV-2) that may be used to differentiate whether a HSV-2 vaccine elicits robust or anemic protection against genital herpes. This gap in knowledge is often attributed to a failure to measure the correct component of the adaptive immune response to HSV-2. However, efforts to identify a correlate of immunity have focused on subunit vaccines that contain less than 3% of HSV-2's 40,000-amino-acid proteome. We were interested to determine if a correlate of immunity might be more readily identified if 1. animals were immunized with a polyvalent immunogen such as a live virus and/or 2. the magnitude of the vaccine-induced immune response was gauged in terms of the IgG antibody response to all of HSV-2's antigens (pan-HSV-2 IgG). Pre-challenge pan-HSV-2 IgG levels and protection against HSV-2 were compared in mice and/or guinea pigs immunized with a gD-2 subunit vaccine, wild-type HSV-2, or one of several attenuated HSV-2 ICP0 (-) viruses (0Δ254, 0Δ810, 0ΔRING, or 0ΔNLS). These six HSV-2 immunogens elicited a wide range of pan-HSV-2 IgG levels spanning an ∼500-fold range. For 5 of the 6 immunogens tested, pre-challenge levels of pan-HSV-2 IgG quantitatively correlated with reductions in HSV-2 challenge virus shedding and increased survival frequency following HSV-2 challenge. Collectively, the results suggest that pan-HSV-2 IgG levels may provide a simple and useful screening tool for evaluating the potential of a HSV-2 vaccine candidate to elicit protection against HSV-2 genital herpes.